Aim: Determine the effect of Anandamide (AEA) and its endogenous precursor 20:4 NAPE (n-arachidonoyl phosphatidylethanolamine; NAPE) on neuronal excitability of dorsal root ganglion (DRG) neurons and pain-like behavior using in vitro and in vivo experiments.Methods: Fluorescence imaging (Fura 2) of calcium changes in cultured DRG neurons with repeated high K+ (30 mM) pulses was used as an in vitro model of neuronal excitability. Intrathecal application of drugs in rats and subsequent measurement of thermal (Hargreaves test) and mechanical sensitivity (Von Frey test) was used to assess nociception.Results: NAPE produced a dose-dependent biphasic response on neuronal excitability. Application of 10nM-1µM NAPE inhibited K+ evoked Ca2+ transients, whereas 10 µM concentration led to potentiation. AEA application followed the same trend. In NAPE-specific phospholipase D inhibitor LEI-401 pre-incubated cells, the changes persisted with AEA but not with NAPE. Intrathecal application of NAPE reduced both mechanical and thermal pain sensitivity with lower doses (2-20 µM ) but produced hypersensitivity at 200 µM. These changes were blunted when LEI-401 was intrathecally applied prior to the NAPE application. Cannabinoid (CB1) receptor inhibition by PF 514273 reduced the inhibitory effect of low-dose NAPE and AEA, whereas excitatory effects were blocked by Vanilloid (TRPV1) receptor antagonist SB 366791.Conclusion: NAPE produced inhibition and excitation of DRG neuronal excitability and spinal cord nociception depending on the concentration, and these effects were mediated by CB1 and TRPV1 receptors, respectively. NAPE in low concentrations may act as a potent inhibitor of nociception.CarDia LX22NPO5104 European Union Next Generation EU